Tumor progression locus 2 (Tpl2)/cancer Osaka thyroid kinase is a newer member of MAP3K family that is now known for its essential role in tumor necrosis factor-aplha (TNFα) expression in macrophages, but its pro-inflammatory signaling, if any, in glia is unknown. When cultures of murine microglia and astrocytes were exposed to lipopolysaccharide, there was a rapid activation (i.e., phosphorylation) of Tpl2 in parallel to the activation of down-stream effector MAPKs, that is, extracellular signal regulated kinase (ERK), p38 MAPK and C-Jun N-terminal kinase (JNK). Pre-incubation of the cultures with a Tpl2 inhibitor selectively suppressed the activation of the primary down-stream target, that is, ERK relative to p38 MAPK and JNK. That Tpl2 activation was functionally involved in glial inflammatory response was indicated by a reduced release of the cytokines, i.e. TNFα and the expression of inducible nitric oxide synthase in the presence of the kinase inhibitor. Furthermore, over-expression of a wild-type Tpl2 construct in C-6 glia resulted in an enhanced transcriptional activation of inducible nitric oxide synthase, while transfection with a dominant negative form of Tpl-2 had the opposite effect. The findings assign an important pro-inflammatory signaling function for Tpl2 pathway in glial cells.
MAP kinase cascades including ERK, JNK, and p38 are involved in glial expression of inflammatory mediators. Recent studies identify TPL2, a newer member of MAP3K, as the major upstream activator of ERK in immune cells although essentially nothing is known about this kinase in glial cells. The studies presented define an important proinflammatory function of TPL2 pathway in these cells.
Inflammatory cell signaling is mainly mediated by coordinated activation of MAP kinase and NFκB pathways in response to a variety of proinflammatory stimuli most commonly represented by the ligands of the Toll-like receptor (TLR) family. Thus, in a simplified scheme, binding of the prototype pathogen, that is, bacterial lipopolysaccharide (LPS) to its receptor TLR4 results in the recruitment of the adapter, MyD88 and formation of a signaling complex comprising the kinases, IRAK1 and IRAK4 and the cytoplasmic adapter TRAF6 (Barton and Medzhitov 2003; Banerjee and Gerondakis 2007; Kawai and Akira 2007; Brikos and O'Neill 2008). This complex engages and activates members of the MAP3K family that in turn form activation nodes for the NFκB and MAPK branches of downstream signaling. Thus, TAK1, a common MAP3K, when activated stimulates IKK complex resulting in the phosphorylation and degradation of the inhibitory IκB proteins thereby releasing and rendering NFκB in its active form that can then translocate to the nucleus for transcriptional activation of target genes (Brikos and O'Neill 2008). TAK1-mediated signaling along the MAPK branch leads to the activation of the effector kinases belonging to the stress-activated protein kinase (SAPK) subgroup, that is, p38 and JNK in a three-tiered kinase cascade (Banerjee and Gerondakis 2007; Kawai and Akira 2007). Specific transcription factors targeted by these MAPKs then coordinate with NFκB and other factors to regulate the expression of immune and inflammatory mediators. We have shown previously that enforced expression of activated TAK1 in glial cells leads to an induction of inflammatory mediator [i.e., inducible nitric oxide synthase, iNOS and IP-10] gene expression and that an inhibition of the down-stream pathways, that is, JNK, p38 and NFκB suppresses this response (Bhat et al. 2003; Shen et al. 2006). In separate studies, we have shown that LPS-induced inflammatory response (i.e., expression of iNOS, cytokines) in glial cells also involves the participation of another effector MAP kinase, that is, extracellular signal-regulated kinase (ERK) (Bhat et al. 1998). ERK is commonly and traditionally known for its role in growth and differentiation responses in non-immune cells where it is activated by receptor Tyr kinases and G protein coupled receptors in a Ras/Raf-dependent way (Chang and Karin 2001). Recent findings indicate that the upstream MAP3K that is solely responsible for ERK activation in response to TLR/IL-1 super family as well as some of the TNF receptor family members is a kinase termed tumor progression locus 2/cancer Osaka thyroid (Tpl2/Cot) (Banerjee and Gerondakis 2007; Vougioukalaki et al. 2011).
Tpl2/Cot was originally identified as a cancer associated gene product (Patriotis et al. 1993; Erny et al. 1996; Ceci et al. 1997) and later characterized as a member (i.e., MAP3K8) of the MAP3K family capable of directly phosphorylating and activating the MAP kinases, that is, MEK and SEK (Salmeron et al. 1996). In unstimulated cells, Tpl2/Cot exists in an inactive state bound to p105 NFκB and ABIN2 (A20-binding inhibitor of NFκB2) among other proteins that protect the kinase from degradation (George and Salmeron 2009; Vougioukalaki et al. 2011). TLR/IL-1R stimulation activates IKKβ kinase that phosphorylates p105 NFκB to trigger its partial degradation to p50NFκB and the release of Tpl2/Cot, which in its phosphorylated state is capable of activating MEK/MKK1/2 before being degraded via the proteasomal pathway. Studies have consistently shown that Tpl2/Cot-mediated ERK activation is essential for LPS-induced TNFα production in macrophages involving post-transcriptional regulation of TNFα mRNA translocation and/or processing of pre-TNFα to its processed mature form (Dumitru et al. 2000; Rousseau et al. 2008; Xiao et al. 2009; Hirata et al. 2010). The kinase has also been shown to regulate secretion of several other cytokines and chemokines in different cell systems (Vougioukalaki et al. 2011). However, essentially nothing is known regarding the activation of Tpl2 in glial cells and its role with respect to neuroinflammation. In this study, using a specific inhibitor and a molecular mutant of the kinase, we show for the first time that Tpl2 signals the release of TNFα and the induction of iNOS gene expression in neuroimmune cells, that is, microglia and astrocytes in response to LPS treatment.
Materials and methods
Astrocyte and microglial cultures
Pregnant mice of C57BL/6 strain were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Primary, mixed glial cell cultures were prepared from the cerebral cortex of newborn mice (both sexes) essentially as described before (Bhat et al. 1998). Briefly, cells isolated from cerebral hemispheres were dissociated in Dulbecco's modified Eagle's medium with 10% calf serum and plated in 75 cm2 culture flasks (Falcon, ThermoFisher Scientific, Waltham, MA, USA) and incubated at 37°C in an atmosphere of 5% CO2 in Air. The medium was changed after 3–4 days and twice a week thereafter. At confluency (12–14 DIV), mixed glial cultures were shaken to dislodge microglia that were loosely attached to the astrocyte monolayer. Microglial cultures were used 24 h after plating. The mixed glia were then shaken overnight to remove oligodendrocyte progenitors and the bed-layer of astrocyte-enriched cultures were subcultured into either 24-well or 6-well plates for various experiments. ARRIVE guidelines were followed up for the use of animals and the procedures were carried out in accordance with the USPHS policy on the Humane Care and Use of Laboratory Animals. The pregnant mice until the delivery were housed in the center for laboratory animals in the Children's Research Institute (7th fl) at MUSC. All animals were fed water and pellets ad libitum and kept on a 12:12 h light/dark cycle and monitored carefully for appearance of diseases. Principles for the Use of Animals and Guides for the Care and Use of Laboratory Animals were strictly followed.
BV2, a mouse microglial cell line, and C6, a rat glial cell line, were maintained and subcultured as needed for experiments as described before (Bhat et al. 2002; Shen et al. 2006).
Enzyme-linked immunosorbent assay
The amounts of TNF-α and IL-1β released into the medium by activated microglia were measured by ELISA performed using ELISA development kits (PeproTech, Rocky Hill, NJ, USA) according to manufacturer's instructions. All samples and standards were assayed in duplicates.
The cells were harvested in lysis buffer (20 mM HEPES [pH 7.8], 350 mM NaCl, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM MgCl2, 20% glycerol, 1% NP-40 with protease inhibitor cocktails I, phosphatase inhibitor cocktail II (Sigma Chem. Co., St. Louis, MO, USA), and 1 mM phenylmethylsulfonyl fluoride) and the protein content determined using the Bradford protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (10 μg) of protein samples were mixed with NuPage Low-density standard sample buffer (4X) (Invitrogen, Carlsbad, CA, USA) with 5% β-mercaptoethanol, heated at 95–100°C and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transblotted onto polyvinylidene difluoride membrane that was subsequently blocked with 5% bovine serum albumin, 1% milk powder in 10 mM Tris-HCl containing 150 mM NaCl and 0.5% Tween 20 (TBST) for 1 h and incubated overnight with appropriately diluted primary antibodies, that is, iNOS (Sigma), IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-p38, phospho-JNK, phospho-ERK, phospho-Tpl2, phospho-Akt (Cell Signaling Tech. Boston, MA, USA) and β-actin (Chemicon, Temecula, CA, USA). Following extensive washing with TBST and incubation with peroxidase-conjugated secondary antibodies, the blots were developed with a commercially available enhanced chemiluminescence reagent.
Total RNA was isolated using Trizol reagent (Invitrogen). The mRNAs were reverse transcribed into cDNAs using M-MLV reverse transcriptase (Invitrogen). Specific primers used for the mouse TNF-α, iNOS and IL-1β were as described (Kitazawa et al. 2005; Thirumangalakudi et al. 2008). Real-time RT-PCR analysis was carried out on a Bio-Rad iCycler system using a SYBR Green qPCR Supermix kit. At the end of the runs, melting curves were obtained to make sure that there are no primer-dimer artifacts. The products were verified by agarose gel analysis. Threshold cycle (Ct) values were calculated with MyiQ software (Bio-Rad) and quantitative fold changes in mRNA were determined as relative to glyceraldehyde 5 phosphate dehydrogenase or 18S mRNA levels in each treatment group.
Transfection and dual luciferase assays
Transient transfections of C6 glioma cultures were carried out using lipofectamine 2000 transfection reagent (Invitrogen). Subconfluent cultures were transfected with plasmids containing WT TPL2, DN (K167N)-TPL2 and empty vector (CMV5) together with pGL3-iNOS firefly Luc reporter plasmid and a plasmid (PRL-TK) expressing the enzyme Renilla luciferase from Renilla reniformis as an internal control. The wtTPL2 and DN-TPL2 were a kind gift from Dr. Christos Tsatsanis, University of Crete, Greece. The iNOS-Luc reporter construct contained a 2168-bp fragment of the rat iNOS promoter with multiple binding sites including cAMP-response element binding protein, C/EBP and NFκΒ cloned into the basic pGL-3 vector (Promega, Madison, WI, USA) (Gavrilyuk et al. 2001; Bhat et al. 2002). After 48 h, the cultures were treated with a combination of LPS (1 μg/mL), Interferon-gamma (IFNγ) (10 ng/mL) and TNFα (10 ng/mL) for 6 h. Firefly and Renilla luciferase activities present in cell lysates were assayed by the use of the Dual Luciferase Reporter System (Promega), with a luminometer as specified by the manufacturer. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as -fold induction relative to control.
Statistical analyses were performed using Statview 5 software (Abacus Concepts Inc., Berkeley, CA, USA). The significance of difference between treated and untreated samples was determined by anova applying Fisher protected least significant difference to get pairwise comparisons among means. The differences were considered significant for the data set with p values < 0.05.
LPS activation of Tpl2 and Tpl2-dependent down-stream signaling in primary glial cells
We first tested if LPS activates Tpl2/Cot in glial cells using a phospho-specific antibody to detect phosphorylated Tpl2 by immunoblot. As shown in Fig. 1a, LPS induced a rapid phosphorylation of the kinase in primary microglia with a peak of activity at 60 min post-treatment. Fig. 1b shows the activation of Tpl2 in astrocytes treated with a combination of LPS and IFNγ, again showing a pattern similar to that in microglia. Densitometric analysis of Tpl2 activation is shown in Fig. 1c.
To test the role of Tpl2 as a mediator of LPS-induced activation of the effector kinases, we used a pharmacological inhibitor of the kinase (Tpl2KI; Calbiochem, San Diego, CA, USA). The kinase inhibitor used is one in a series of cell-permeable naphthyridine compounds that has been shown to act as a potent, reversible, and ATP-competitive inhibitor of Tpl2 kinase with significant selectivity over other related kinases including epidermal growth factor receptor, MEK, MK2, p38, Src, and protein kinase (Gavrin et al. 2005). These compounds (or their relatives) have been shown to inhibit LPS-induced TNFα production from human monocytes in vitro (Gavrin et al. 2005) and in LPS/D-Gal-induced mouse model of rheumatoid arthritis (Hu et al. 2006). A time course of the activation/phosphorylation of ERK, JNK and p38 in microglia treated with LPS in the presence and absence of the Tpl2KI is shown in Fig. 2. It is clear that an inhibition of Tpl2 most effectively suppresses the activation of ERK in agreement with Tpl2-dependent activation of ERK in other systems. On the other hand, the activation of p38 kinase and JNK was inhibited to a lesser extent. To test if Tpl2 is linked to NFκB pathway, we evaluated IκB degradation and NFκB-dependent transcriptional response (NFκB-Luc reporter activity) in LPS-treated microglia and astrocyte cultures in the presence and absence of Tpl2KI. The results (not included) showed minimal/no changes indicating a lack of Tpl2-dependent NFκB activation.
Figure 3 illustrates the effects of Tpl2 inhibition on LPS activation of the down-stream MAP kinases in astrocytes. The results (-densitometric analyses shown in Fig. 3b) confirm preferential activation of ERK by Tpl2 signal in astrocytes as in microglia.
The role of Tpl2 in LPS-induced expression of TNF∝ and iNOS in glial cells
A well-described role of Tpl2 in macrophages and other immune regulatory cells is its stimulation of TNFα expression in response to LPS. That Tpl2 also regulates TNFα release in microglia is indicated by data included in Fig. 4. Thus, the kinase inhibitor suppresses the secretion of TNFα in cultures treated with LPS with or without IFNγ. The Tpl2 inhibitor had a similar inhibitory effect on NO release in LPS-treated microglia (Fig. 5a) and in LPS/IFNγ-treated astrocytes (Fig. 6a). The changes in NO levels corresponded to the expression levels of iNOS (Figs 5b and 6b). It should be noted that the kinase inhibitor exerted minimal toxicity toward the cells even at the highest dose (5 μΜ) used, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
We next determined the effects of Tpl2 inhibition on the expression levels of iNOS and TNFα mRNA in cultures of a mouse microglia cell line, BV2 treated with LPS. As shown in Fig. 7a, inhibition of Tpl2 resulted in reduced levels of iNOS mRNA without any effect on TNFα mRNA in agreement with reported post-transcriptional control of TNFα expression. However, the Tpl2 inhibitor blocked the expression of another cytokine, IL-1β at the transcriptional level thereby indicating multiple ways that Tpl2 may regulate the expression of individual mediators. Fig. 7b and c show parallel expression of iNOS and the cytokines at the protein level as determined by western blot and ELISA, respectively. In general, we have observed that Tpl2 blockade results in a more robust inhibition of iNOS protein rather than iNOS mRNA in line with multiple mechanisms of Tpl2 regulation of its target genes.
Molecular evidence for the role of Tpl2 in iNOS gene expression
To further implicate the role of Tpl2 in glial inflammatory response we used a molecular approach, that is, the consequence of transient transfection with Tpl2 mutants on the transcriptional activation of iNOS gene. Cultures of a glial cell line, C-6 were transfected with either the wild-type Tpl2 or its dominant negative form along with iNOS-luc. Sets of cultures were then treated with a combination of LPS, IFNγ and TNFα (LTI), a regimen used before to induce iNOS promoter in this cell system (Bhat et al. 2002) and assayed for changes in the activity of iNOS-luc. As shown in Fig. 8, there was an increased iNOS-luc activity in cells transfected with the Wt Tpl2 over control (empty vector) that was further enhanced (twofold) by treatment with the cytokine combination. In contrast, cells transfected with DN Tpl2 plasmid expressed reduced level of iNOS-luc activity that was only minimally enhanced in response to cytokine treatment.
In this study, we show that the activation of Tpl2 kinase acts as an essential signal for the expression of proinflammatory mediators in glial cells in response to LPS stimulation. The results are in agreement with such a proinflammatory function ascribed to this kinase pathway in other cell systems, that is, peripheral macrophages (Dumitru et al. 2000), adipocytes (Jager et al. 2010), hepatic stellate cells (Perugorria et al. 2013), and airway epithelial cells (Martel et al. 2013) as well as in animal models of inflammation [reviewed in (Vougioukalaki et al. 2011)]. With respect to Tpl2-mediated signaling pathways in glia, our results with a chemical inhibitor of the kinase suggest Tpl2-dependent activation of down-stream effector kinases, in particular, ERK that likely mediate induced expression of iNOS and proinflammatory cytokines, that is, TNFα and IL-β. As in other systems, the kinase shows versatility in its regulation of target gene expression at both transcriptional (i.e., iNOS, IL-β) and post-transcriptional/translational (i.e., TNFα) levels. Although not tested in this study, the mechanism by which Tpl2 regulates post-transcriptional expression of TNFα may involve mRNA translation, translocation and/or ERK-induced activation of TNFα converting enzyme as in macrophages (Rousseau et al. 2008; Hirata et al. 2010; Lopez-Pelaez et al. 2012). Our previous studies have indicated a similar differential (i.e., transcriptional vs. post-transcriptional) regulation of TNFα vs. iNOS expression in glial cells in a p38/ERK MAP kinase-dependent mechanism (Bhat et al. 1998). This study extends these observations to include Tpl2 as an upstream mediator of LPS/TLR4 signal that may primarily target ERK, while other MAP3Ks including TAK1 preferentially activate the SAPK subgroup of MAP kinases. Thus, it is clear that Tpl2-activated ERK, in concert with TAK1-induced JNK/p38 and NFκB branches of the MAPK cascade, mediates an efficient induction of the full array of TLR targets in glia as well as in other immune-regulatory cells.
The results of our transient transfection studies with plasmid vectors expressing the wild type and a DN form of Tpl2 further support Tpl2-mediated transcriptional induction of iNOS in glia. However, these findings seem at odds with a recent report that suggested a repressive function for Tpl2 signal in iNOS gene expression in macrophages (Lopez-Pelaez et al. 2011). Thus, macrophages derived from Tpl2-null mice when treated with LPS responded with an induction of iNOS expression. These cells also display a reduced activation of PI3K-Akt, a pathway known to keep iNOS expression in check in TLR-activated macrophages (Diaz-Guerra et al. 1999). In the light of these findings, we tested if Tpl2 inhibition has any effect on LPS-induced Akt phosphorylation in glial cells. However, we did not see any effect of the kinase inhibitor on the phosphorylation status of Akt in LPS-activated (or untreated control) glial cells (data not shown). The contradictory findings on the role of Tpl2 in iNOS expression in macrophages versus glial cells may relate to cell type differences and/or distinct experimental approaches, that is, pharmacological or molecular inhibition of the kinase (present study) versus genetic deletion of the kinase [Lopez-Palaez et al. (Lopez-Pelaez et al. 2011) study].
There is increasing evidence that Tpl2 pathway mediates inflammation in a number of peripheral diseases. Thus, in vivo studies with mice deficient in Tpl2/Cot have confirmed its proinflammatory role in septic shock (Dumitru et al. 2000), inflammatory bowel disease, periodontitis, severe acute pancreatitis, pathogen infection, zymosan-induced inflammatory nociception (Soria-Castro et al. 2010), and other diseases/conditions involving peripheral inflammation (Vougioukalaki et al. 2011). Being an upstream kinase in the MAPK cascade that specifically signals the activation of ERK in activated immune cells, Tpl2 represents an attractive target for anti-inflammatory intervention (George and Salmeron 2009). A unique advantage of this approach is that Tpl2 inhibition spares the activation of ERK1/2 by other MAP3Ks including Raf1 thereby preserving ERK-dependent physiological functions. It is also likely that targeting of an upstream kinase such as Tpl2 in a signaling cascade may turn out to be more effective than blocking individual down-stream kinases. In support of this idea, the results of our studies show that an inhibition of Tpl2 is more effective in suppressing iNOS expression (potentially involving both transcriptional and translational regulatory mechanisms) than inhibition of either ERK or p38 MAPK alone (Bhat et al. 1998).
In conclusion, the present demonstration of the proinflammatory signaling function of Tpl2 in glia prompts further investigation into its potential role in neuroinflammation associated with neurodegenerative and neuroinfectious conditions and as a novel anti-inflammatory treatment target.
The Tpl2 expression plasmids were a gift from Dr Christos Tsatsanis, University of Crete, Greece. The study was supported in part by NIH grant R21NS063183 and by the National Center for Research Resources through grant number C06 RR015455. The authors declare that they have no conflict of interest regarding the work presented here.